Method and Kit for Identifying Gene Mutations

ABSTRACT

This invention relates to a method of identifying mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a kit for performing the method, and furthermore to isolated nucleotide sequences being complementary to one or more mutations of the CFTR gene. According to a first aspect of the invention there is provided a method of identifying mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, including the steps of providing one or more nucleic acid sequences, fully complementary to one or more segments of the CFTR gene, wherein the one or more nucleic acid sequences correspond to the mutation to be identified; providing a biological sample of an individual to be tested for CF; isolating nucleic acids from the biological sample; and testing the biological sample for the presence of one or more of the nucleic acid sequences using a suitable detection method.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/773,800, filed May 4, 2018, now U.S. Pat. No. 11,060,146; whichis a National Stage Application of International Application NumberPCT/IB2016/056606, filed Nov. 3, 2016; which claims priority to GreatBritain Application No. 1519501.9, filed Nov. 4, 2015.

The Sequence Listing for this application is labeled“SeqList-04May18-ST25.txt”, which was created on May 4, 2018, and is 7KB. The entire content is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to a method of identifying mutations in thecystic fibrosis transmembrane conductance regulator (CFTR) gene, a kitfor performing said method, and furthermore to isolated nucleotidesequences being complementary to one or more mutations of the CFTR gene.More particularly, but not exclusively, the invention relates to amethod and kit identifying mutations in the cystic fibrosistransmembrane conductance regulator (CFTR) gene in respect of patientsof African origin, and specifically black and mixed raced patients.

BACKGROUND TO THE INVENTION

Cystic fibrosis (CF) is the most common, potentially lethal autosomalrecessive disease among individuals of Caucasian descent in the world.CF is caused by mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) gene, an ion channel protein primarilyresponsible for the trans-epithelial conductance of chloride ions.Dysfunctions in this protein lead to various symptoms being observed inCF patients, with the most common being the so called classic triad ofCF symptoms, being elevated sweat chloride concentration, pancreaticinsufficiency and chronic pulmonary disease, although this classic triaddoes not necessarily present in all CF patients, which could lead tomisdiagnosis. Associated CF symptoms include failure to thrive, maleinfertility and microbial colonisation of the airway.

Whilst CF amongst patients of Caucasian descent has been studied anddocumented relatively extensively, CF is not limited to this demographicgroup, and has been identified in blacks and individuals of mixed raceancestry. This bias has affected diagnosis of, and research in,non-white patients, because of the underlying assumption that thisdisease could not affect other racial groups. As a result, Europeanpopulations tend to have the highest rates of mutation detection andmolecular diagnosis.

Literature indicates that CF patients from African (black and mixedrace) ancestry are at a distinct disadvantage when compared to theirEuropean counterparts. The data suggests that the number of CF causingmutations in patients of European descent may be approaching a plateau,while the opposite is true for African CF patients.

There are two major challenges to diagnosing CF in African CF patients.First, they have a relatively rare disease which occurs at lowerfrequencies than those seen in patients of European descent. This mayincrease their chances of being misdiagnosed especially in areas wherethere are more rampant phenocopic illnesses such as malnutrition, viralor parasitic infection or tuberculosis. Second, there is not enoughinformation available for the design of Afro-centric genetic tests. Thisincreases the probability of misdiagnosis particularly if the patientsdon't present with the classic triad of CF symptoms or if they havemilder forms of the disease. It is therefore important to cease theexclusion of CF as a diagnosis based on race.

U.S. Pat. No. 8,338,578 describes novel mutations of the CFTR generelated to CF or to conditions associated with CF, and further to probesfor detecting mutant CFTR sequences, and methods of identifying saidmutations of the CFTR gene in the genotype of an individual. However, italludes to CF being the most common severe autosomal recessive geneticdisorder in the Caucasian population, adding to the common misconceptionthat CF does not affect patients of black and mixed race ethnic origins.

OBJECT OF THE INVENTION

It is accordingly an object of the invention to provide a method foridentifying mutations in the cystic fibrosis transmembrane conductanceregulator (CFTR) gene in respect of patients of African origin, and inparticular black and mixed race patients, but without limiting themethod to only these groups of patients, and a diagnostic kit forperforming said method.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a methodof identifying mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) gene, including the steps of:

-   -   providing one or more nucleic acid sequences, fully        complementary to one or more segments of the CFTR gene, wherein        the said one or more nucleic acid sequences correspond to the        mutation to be identified and may be selected from the group        comprising SEQ ID: 1-3;    -   providing a biological sample of an individual to be tested for        CF;    -   isolating nucleic acids from the biological sample; and    -   testing the biological sample for the presence of one or more of        the nucleic acid sequences using a suitable detection method.

The biological sample may be any sample obtained from the individual'sblood, serum, plasma, urine, skin, hair or any other biological samplecontaining DNA.

Further according to a first aspect of the invention, the detectionmethod may be one or more methods selected from the group consisting ofan amplification refractory mutation system (ARMS), next generationsequencing (NGS), quantitative polymerase chain reaction (qPCR) andmicroarrays.

Still further according to a first aspect of the invention, the ARMSdetection method may comprise one or more tetra-ARMS primers selectedfrom the group consisting of SEQ ID: 4-15. The NGS detection method maycomprise one or more probes selected from the group consisting of SEQID: 16-21. The qPCR detection method may comprise one or more primersand probes selected from the group consisting of SEQ ID: 22-27, as wellas the corresponding hybprobes selected from the group consisting of SEQID: 28-33. The microarray detection method may comprise one or moreprobes selected from the group consisting of SEQ ID: 34-36.

Yet further according to the invention, the individual may be of Africanorigin, and may more specifically be a black or mixed raced individual.

In a second aspect of the invention there is provided a method fordiagnosing cystic fibrosis in an individual comprising the steps of:

-   -   obtaining a biological sample from the individual;    -   isolating nucleic acids from the sample; and    -   testing the biological sample for the presence of one or more        nucleic acid sequences, fully complementary to one or more        segments of the CFTR gene, wherein said one or more nucleic acid        sequences correspond to the mutation to be identified and may be        selected from the group comprising SEQ IDs: 1-3.

According to a third aspect of the invention there is provided a methodfor determining if an individual or the individual's offspring will havea predisposition to CF, the method comprising the steps of:

obtaining a biological sample from the individual;

isolating nucleic acids from the sample;

testing the biological sample for the presence of one or more nucleicacid sequences, fully complementary to one or more segments of the CFTRgene, wherein said one or more nucleic acid sequences correspond to themutation to be identified and may be selected from the group comprisingSEQ IDs: 1-3; and

-   -   determining from the test results if the individual or the        individual's offspring has a predisposition for CF, and advising        the individual accordingly.

According to a fourth aspect of the invention there is provided a kitfor performing one or more of the methods according to the invention. Inparticular, the kit may be provided with primers and probes required todetect the presence of nucleic acids using the detection methods inaccordance with one or more of the aspects of the invention.

According to a fifth aspect of the invention there is provided one ormore nucleic acid sequences, fully complementary to one or more segmentsof the CFTR gene, wherein said one or more nucleic acid sequencescorrespond to the mutation to be identified and may be selected from thegroup comprising SEQ IDs: 1-3.

Further according to any of the aspects according to the invention, theinvention can be used to detect CF, but also CF related diseases orother monogenic disorders.

Further according to any of the aspects according to the invention, theinvention may be used in the detection of complex alleles consisting oftwo or more CFTR gene mutations.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

In accordance with the invention, cystic fibrosis transmembraneconductance regulator (CFTR) gene mutations are identified by aplurality of steps.

DNA Isolation

In a first step, one or more isolated purified nucleic acids eachcomprising 25 nucleotides occurring in the CFTR DNA, the nucleic acidsbeing fully complementary to one or more segments of the CFTR gene, areselected from the group of CFTR gene mutations comprisingc.1277_1278insAT, c.3512_3516dupCAGAA or c.2630delC.

Blood samples are collected from patients that are believed to besuffering from CF, and DNA is isolated from the blood of the patients,whereafter the presence of one or more of the CFTR gene mutations in theDNA is determined using a suitable detection method.

The protocol for extracting the DNA from the blood samples is initiatedonce the white blood cell count (WBCC) for a particular patient isdetermined, which usually occurs within 24 hours of a patient giving ablood sample. A maximum of 2×10⁷ white blood cells are recommended percolumn for spin-column based nucleic acid purification (in thisembodiment, the QIAGEN column is used). Samples containing more thanthis can be diluted using 1× phosphate buffered saline (PBS) and equalvolumes applied to two separate columns.

In this embodiment, QIAGEN protease (200 μL) is then placed in thebottom of a 15 mL centrifuge tube, 2 mL of blood added and the tubesubjected to a brief vortexing step to ensure thorough mixing. Buffer AL(2.4 mL) is added to the mixture, the tube inverted 15 times and thenshaken vigorously by hand for 1 minute, before being incubated at 70° C.for 10 minutes. The tube is removed, 2 mL of molecular biology gradeabsolute ethanol added, the tube inverted 10 times and shaken vigorouslyby hand for a minute. Half of the solution is transferred to a QIAampMidi column in a 15 mL centrifuge tube without moistening the rim. Thetube is spun at 1850 g for 3 minutes, the column removed, the filtratediscarded, the remainder of the solution applied to the column and thecentrifugation step repeated. Without touching the rim of the column, 2mL of Buffer AW1 is added and the closed tube spun at 4500 g for 1minute. Again without moistening the rim, 2 mL Buffer AW2 is added, thetube closed and spun at 4500 g for 15 minutes. The filtrate isdiscarded, the column placed in a new 15 mL tube and 300 μL roomtemperature Buffer AE added to the centre of the column. The tube isthen incubated at room temperature for 5 minutes and spun at 4500 g for2 minutes. The resulting eluate is removed from the tube, placed in thecentre of the column and the procedure repeated to obtain a moreconcentrated first eluate. The column is then placed in a new 15 mLtube, an additional 300 μL room temperature Buffer AE added to thecentre of the column and the elution steps above repeated to obtain asecond eluate.

A spectrophotometer (such as the NanoDrop spectrophotometer) is used forDNA quantification purposes. A 1:10 dilution of the first eluate is madeusing water as the diluent; the second eluate is then read undiluted.Buffer AE (2 μL) is used as the blank. The instrument is re-blankedafter every three to four readings, and each sample is measured using 2μL of either the diluted (first eluate) or undiluted (second eluate)DNA.

Each eluate (5 μL) is then visualised on a 0.7% agarose gelelectrophoresed in 1XTBE (tris-borate-EDTA), stained with nucleic acidstain (such as Biotium GelRed) diluted 1:16,667 times in 50 mL of moltenagarose. The DNA is then subjected to electrophoresis at 90 V of currentusing Lab Aid Mass Ruler High Range as the molecular ladder.

Mutation Detection

Suitable detection methods for determining the presence of CFTR genemutations include the amplification refractory mutation system (ARMS),next generation sequencing (NGS), quantitative polymerase chain reaction(qPCR) and microarrays.

The amplification refractory mutation system (ARMS) can be used indiagnosing various diseases including cystic fibrosis (CF). Whereas ARMSrequires separate reactions to identify the mutant and the wildtypesequences, tetra-primer ARMS allows for the identification of bothmutant and wildtype in the same reaction tube relying on the use of fourprimers. The first primer pair are the outer flanking primers which arebased on the wildtype sequence and there is a second pair of innerprimers. The forward inner primer is specific to the wildtype sequencewhereas the reverse inner primer is specific to the mutant sequence (Yeet al., 2001; Ye et al 1992). This methodology can be used to identifythe mutations discovered in CF patients in general, but moreparticularly in non-white CF patients, with the relevant primers,corresponding to SEQ ID: 4-15, being listed in table 1. In addition tothese primers, the reaction requires a suitable PCR reaction buffer, TaqDNA polymerase, deoxyribose nucleoside triphosphates (dNTP's),nuclease-free water, and the patient DNA. This is subjected to either anamplification program involving five cycles at the annealing temperatureof the flanking primers, followed by 30 cycles at the annealingtemperature suitable for the inner primers or a touchdown PCR with theinitial annealing temperature being 72° C., decreasing by 1° C. percycle until the temperature of the inner primers is reached andcontinuing at that temperature until the end of the PCR programme. Thegeneral programme also has an initial denaturation step at 95° C. for 1minute. The amplification step consists of 35 cycles of 95° C. for oneminute, one minute at the previously described annealing temperature andone minute at 72° C. There will be a final extension step at 72° C. forthree minutes. The amplicons are then resolved on a 1% agarose gel. Theexpected amplicon sizes are also listed in table 1.

TABLE 1 The tetra-ARMS primers for identification of CF mutations.Annealing Amplicon Mutation Primers Temperature/° C. size/bp SEQ ID: 1ACAATAGAAAAACTTCTAATG 58.66 Wt: 409 GTGATAAC (fi) Mt: 223GAGAAATTACTGAAGAAGAGG 60.89 CTGATT (ri) CAGTGTAATGGATCATGGGCC 72.66ATGTGCT (fo) GCTCGCCATGTGCAAGATACA 70.8 GTGTTG (ro) SEQ ID: 2CCATTCCAGGTGGCTGCCTC 64.82 Wt: 345 (fi) Mt: 188 GGAGCCACAGCACAACCACAA63.92 (ri) CTGCTGGACCCAGGAACAAAG 73.65 CAAAGG (fo) CACTATATTGTCCAGGCTGGA73.4 GTGCGGTG (ro) SEQ ID: 3 TTCATTGACATGCCAACAGTA 56.25 Wt: 299 G (fi)Mt: 447 GGTAGGTTTACCTTCTGTTCTG 56.23 TT (ri) GAATCTTCAGTAGTGGTTTTGA66.99 GGTGTGG (fo) TGCTAACACATTGCTCAGGCT 69.74 ACTGGG (ro) Fi = forwardinner; fo = forward outer; ro = reverse outer; ri = reverse inner; wt= wildtype; mt = mutant

In accordance with the invention, next generation sequencing (NGS) couldalso be used to identify the CF mutations in a sample of patient DNA.Probes specific to these mutations are used to assist in diagnosingpatients carrying these variants. A pair of probes, one annealingupstream of the mutation and the other annealing downstream, is neededper mutation. A DNA polymerase is used to fill in the gap between theprobes with complementary dNTPs. Ligase is then used to join theneighbouring bases, resulting in a complete double stranded molecule.Once double-stranded DNA has been produced by these processes, PCR isused to add sequencing primers and indices to the construct. The libraryof DNA fragments bound to the probes are then ready for NGS, which is anautomated process. The applicable probes, corresponding to SEQ ID:16-21, are listed in table 2.

TABLE 2 Probes that are used to identify CFTR mutations via NGS.Mutation Upstream Probe Downstream Probe SEQ ID: 1 GTGTGTTTTTTTAACAGGGGGATCCAGCAACCGCCAACAACTG ATTTGGGGAAT SEQ ID: 2 ATGTGAATTTAGATGTGGGTCTTTCCACTACCATAATGCTTGGG CATGGGAG AG SEQ ID: 3 GCCCGACAAATAACCAAGTAGGGCCAGATGTCATCTTTCTTCAC GACAAATAGC G

Quantitative PCR (qPCR) is an analytical technique that allows thereal-time tracking of the process of amplification. qPCR hybprobes, inconjunction with qPCR primers, can be used to ascertain if the mutationsdescribed are in fact present in a sample of human DNA. The necessaryprimers and probes, corresponding to SEQ ID: 22-33, are listed in table3. In addition to the primer and probe pairs, the reaction also needs toinclude an appropriate qPCR master mix and about 250 ng of template DNA.The probes are labelled with the fluorophores listed in order to allowfor detection of the mutations, if present, while the amplification isproceeding. The reactions could be further optimised to allow formultiplexing to enable all three mutations to be assayed forsimultaneously in one reaction vessel. The amplification programmeconsists of one cycle at 94° C. for 3 minutes, thirty cycles of 94° C.for 30 seconds, 55° C. for 30 seconds and 72° C. for 30 seconds. Thefluorescence is then captured at the annealing step. After thecompletion of the amplification, a melt curve analysis is used todistinguish between the wildtype and mutant sequences. This is executedby heating the reaction tubes to 95° C. for 30 seconds, lowering thetemperature to 45° C. and then raising the temperature to 95° C. in 0.1°C. increments with fluorescence being continuously captured as thetemperature is increased.

TABLE 3qPCR primers and probes utilised to identify CFTR gene mutations inhuman DNA samples Mutation qPCR Primers Hybprobes SEQ ID: 1F: ATGGGCCATGTGCTTTTCAAAC TGATGAATCAGCCTCTTCTTCA R: GCAACCGCCAACAACTGTCCGTAATTTCTC-Fluroscein Texas Red- ACTTCTTGGTACTCCTGTCCTGAAAGATATTAATT-PHO SEQ ID: 2 F: TAGATGTGGGCATGGGAGGATTTTTGGTTGTGCTGTGGCTCC R: CCACTACCATAATGCTTGGGAGA TT-Fluorescein Cy5-GGAAAGTGAGTATTCCATGTC CTATTGTGTAG-PHO SEQ ID: 3F: GCCCGACAAATAACCAAGTGAC GCCAACAGAACAGAAGGTAAA R: ACAGTCATTTGGCCCCCTGCCTACCAAGT-Fluorescein LCRed640- CAACCAAACCATACAAGAATG GCCAAC-PHO PHO= phosphorylated 3′ end

Microarrays are based on the principle of hybridisation. DNA fragments(probes) are attached to a solid substrate (such as a chip) andimmobilised. Nucleic acid from a relevant source is denatured, labelledand allowed to incubate with the probes at an appropriate temperatureovernight. The substrate is then washed to remove unbound DNA and theformation of bonds between the probes and the target DNA detected usinga method appropriate for the label that is used in the beginning of theprotocol. Probes that could be used as part of a microarray screeningfor CF-associated mutations, corresponding to SEQ ID: 34-36 are listedin table 4. The genomic DNA is denatured, snap cooled on ice andlabelled dNTPs and Klenow fragments used to incorporate labels into thesequence. The labelled DNA is purified using a PCR purification kit suchas the QIAQuick PCR purification kit. The hybridisation andvisualisation protocol for the commercially available microarrayselected is followed, and involves a pre-hybridisation step involving abuffer and the chip at a suitable temperature. The labelled DNA is thenintroduced to the microarray chip and incubated overnight at thepre-hybridisation temperature. The chip is washed using solutionscontaining SDS (sodium dodecyl sulphate) and SSC (sodium citrate andsodium chloride, pH 7). Hybridisation is detected as prescribed for thesystem used.

TABLE 4 The microarray probes for CF mutation detection MutationMicorarray Probe SEQ ID: 1 ATGGTGATGAATCAGCCTCTTCTTC SEQ ID: 2AGGTGGCTGCTTTTTGGTTGTGCTG SEQ ID: 3 ACATGCCAACAGAACAGAAGGTAAA

Bioinformatics Pipeline for the Detection of Variants in Human DNA

The initial analyses (including base calling and extracting clusterintensities) are conducted using real-time analysis software suite, suchas Illumina MiSeq RTA 1.14.23. Here a sequence quality filtering scriptis executed using Illumina CASAVA version 1.8.2. Data analysis isconducted in four stages. In the first stage, CASAVA's variant calls areassessed for novelty and potential functional consequences usingsuitable analysis approaches, as known in the art. Here the onlineVariant Effect Predictor tool(http://www.ensembl.org/info/docs/tools/vep/index.html) from Ensembl isused. This tool is asked to return SIFT and PolyPhen scores as well asco-located variations and information, if available, about each variantpresent in the 1000 Genomes Project.

Second, the raw sequence files are imported into a suitable genomicanalysis software suite where they are assessed for quality. Sections ofthe sequence that fall below a quality score of 20 are trimmed, as areany remaining adapter sequences identified. The reads are mapped tochromosome seven and CFTR (version hg19; both obtainable from the onlineUCSC Genome browser database:http://hgdownload.soe.ucsc.edu/downloads.html) and quality based variantdetection used to identify differences between the reads and thereference sequences. Only variants present in both forward and reversereads are returned. The variants called after being mapped to CFTR areannotated with exon number, possible changes to the amino acid sequenceand possible splice site effects; those called after being mapped tochromosome seven are annotated from known dbSNP variants and withconservation scores based on the chromosome seven PhastCons wiggle fileobtained from the UCSC Genome browser. Potential structural variants andindels are also identified.

Third, a bioinformatics pipeline is constructed using existingbioinformatics approaches as known in the art. The raw files areassessed for quality and sections of sequence with a quality score lessthan 20 are trimmed. The trimmed (as needed) raw sequence data, the*.vcf files from both CASAVA and the CLC Genomics Workbench processes,the dbSNP database, the Mills and 1000 Genomes known indels database,the CFTR sequence and the human genome (version hg19) are used as inputdata in the bioinformatics pipeline. A sequence alignment tool, as knownin the art, is used to map the reads to the human genome, whilestatistical approaches, as known in the art, are used to report on thestatistics of the mapping (such as the percentage of reads mapped to thereference). Any alignment discrepancies generated by the presence ofindels are located and these errors fixed. The quality scores for eachread position are then corrected, taking into account errors inherent inthe NGS technology while preserving biologically known variants.Genotyping tools and approaches, as known in the art, are used to callvariants, which are then filtered and annotated. The output of thebioinformatics pipeline thus contains an annotated list of variantsidentified by the various software tools used during the analysis. Thisanalysis is done for each participant and the data exported as separatefiles.

All the variants from all the study participants are copied into onespreadsheet. Conditional formatting is used to identify the duplicatevariants which are removed. These unique calls are manually edited tothe input format required for additional annotation. The data returnedafter annotation is downloaded into a spreadsheet and the variantsfiltered by consequence. The data must be closely examined, payingparticular attention to whether or not the variant has already beenidentified, whether or not the global minor allele frequency and theminor allele frequency per population is <1% (infrequent CFTR variantsare more likely to be associated with pathology; Raynal et al., 2013;Bombieri et al., 2000), which exon or intron it is located in, whetheror not its clinical significance is known, and its HGVSc and HGVSpnotations. Exonic variants are additionally examined for their SIFT andPolyPhen scores. They may also assessed by means of further softwaretools designed to facilitate integration of computational tool output.Here the software tool Condel (González-Pérez and López-Bigas 2011) isused. Condel takes the weighted average of the normalised scores of fivein silico predictive tools (including SIFT and PolyPhen) resulting in agreater degree of accuracy. All the intronic variants are formatted asrequired before being uploaded to the online software tool RegulomeDB(http://regulome.stanford.edu/; Boyle et al, 2012) which utilises ENCODEand other data to rank intronic variants by their likely impact ontranscription and gene regulation. Variants which are identified ashaving an impact on the splice sites of the CFTR gene are subject toadditional analyses using the software tools Human Splicing Finder 2.4.1(http://www.umd.be/HSF/HSF.html; Desmet et al 2009) and SpliceAid2(http://193.206.120.249/splicing_tissue.html; Piva et al 2012).Additionally, all identified variants must be checked against the CFTR2database (Sosnay et al, 2013; http://cftr2.org/mutations_history.php) inorder to find out if their functional significance has already beenempirically determined. These data, in addition to the patient'sclinical file, are used to determine which variants might be potentiallypathogenic.

Fourth, programs are used to provide a level of in silico validation ofthe aligned files respectively. Here pibase (Forster et al., 2013) andBAYSIC (Cantarel et al., 2014) are used. Since both programs werewritten for use in a command line environment, the operating systemubuntu 12.04 (Precise Pangolin) is installed. The programming languagesPython 2.7.3 and pysam 0.6 are installed as pre-requisites for usingpibase. The Python path is exported before using pibase every time a newterminal is opened. A file generated by the bioinfounatics pipeline issorted, MD-tagged and indexed. From within the pibase directory, data isextracted from this file at positions specified by the file outputgenerated by the bioinformatics pipeline using all the variant callingprograms. The command defaults are used except the read length, whichfor the current data is 150 bp; the chromosome naming convention used inthe files need to be identical. A script is used to determine what thebest genotype at the positions of interest are. Since pibase ignoresindels, any position of interest where more than three reads are ignoredis examined using a genome visualisation tool, as known in the art, toallow for manual inspection of the region of interest.

The Tabix and vcftools are compiled from within the BAYSIC directory.The posterior probability for each variant called by the variant callersis calculated from the BAYSIC directory using the correct commands, withthe names of the input files which are downloaded from thebioinformatics pipeline before the filtering and annotation steps.

CONCLUSION

Resolving a patient's molecular diagnosis in this way would also beuseful in designing a genetic test with a higher mutation detection rateand in deciding if a patient may benefit from CF class specific drugs.This should prove especially useful for populations with high geneticdiversity, such as individuals of African descent, which have sufferedfrom the inherent European bias in the current genetic tests. Sequencingcoupled with an appropriate public health policy could lower the age atdiagnosis for CF patients which should both decrease morbidity and raiselife expectancy. This unbiased approach would assist in graduallyeliminating the immediate diagnostic disadvantage faced by non-CaucasianCF patients.

It is accordingly asserted that the disadvantages associated with knownmethodologies and diagnostic practises for determining and confirming adiagnosis of CF, and in particular in individuals of African or mixedrace origin, could be alleviated with the methodology and kit accordingto the invention.

In particular, the disadvantage of having to rely solely on knowndiagnostic methods, including the so called classic triad of symptoms,is overcome by providing a molecular basis for determining and/orconfirming a diagnosis of CF in patients of African or mixed raceorigin, where this molecular basis had not previously been available.

The disadvantage of a diagnostic bias existing in favour of themolecular diagnosis of CF in Caucasian patients is alleviated by themethod and kit according to the invention providing a viable moleculardiagnosis of CF in patients of African or mixed race origin by theutilisation of a method to identify CFTR gene mutations that have notpreviously been documented, and which mutations are evident in patientsof African and mixed race origin.

Accordingly, the perceived and internationally prevalent misconceptionthat patients of African or mixed race origin are not susceptible to CFis disproven in accordance with the invention, which should ultimatelybenefit African or mixed race individuals that suffer from CF. It willfurther increase the overall positive diagnosis of CF in individuals ofany origin, in view of the methodology according to the invention beingfocussed on but not limited to CF patients of African and mixed raceorigin, and accordingly includes any other patients presenting the CFTRgene mutations described herein.

It will be appreciated that in terms of the invention, variations indetails are possible without departing from the scope of this disclosureand the appended claims.

1. A method of identifying mutations in the cystic fibrosistransmembrane conductance regulator (CFTR) gene of an individual,including the steps of: providing one or more nucleic acid sequencesfully complementary to one or more segments of the CFTR gene, whereinthe said one or more nucleic acid sequences corresponds to the mutationto be identified and is selected from the group consisting of SEQ IDNOs: 1-3; providing a biological sample of the individual to be testedfor CF; isolating nucleic acids from the biological sample; and testingthe biological sample for the presence of one or more of the nucleicacid sequences using a suitable detection method.
 2. The method ofidentifying mutations in the CFTR gene of an individual according toclaim 1, wherein the detection method utilizes one or more methodsselected from the group consisting of an amplification refractorymutation system (ARMS), next generation sequencing (NGS), quantitativepolymerase chain reaction (qPCR) and microarrays.
 3. The method ofidentifying mutations in the CFTR gene of an individual according toclaim 2, wherein the ARMS detection method comprises one or moretetra-ARMS primers selected from the group consisting of SEQ ID NOs:4-15.
 4. The method of identifying mutations in the CFTR gene of anindividual according to claim 2, wherein the NGS detection methodcomprises one or more probes selected from the group consisting of SEQID NOs: 16-21.
 5. The method of identifying mutations in the CFTR geneof an individual according to claim 2, wherein the qPCR detection methodcomprises the use of one or more primers and probes selected from thegroup consisting of SEQ ID NOs: 22-27, as well as the correspondinghybprobes selected from the group consisting of SEQ ID NOs: 28-33. 6.The method of identifying mutations in the CFTR gene of an individualaccording to claim 2, wherein the microarray detection method comprisesthe use of one or more probes selected from the group consisting of SEQID NOs: 34-36.
 7. A method for diagnosing cystic fibrosis (CF) in anindividual comprising the steps of: obtaining a biological sample fromthe individual; isolating nucleic acids from the sample; and testing thebiological sample for the presence of one or more nucleic acid sequencesfully complementary to one or more segments of the CFTR gene, whereinsaid one or more nucleic acid sequences corresponds to the mutation tobe identified and is selected from the group consisting of SEQ ID NOs:1-3.
 8. A method for determining if an individual or the individual'soffspring will have a predisposition to CF, the method comprising thesteps of: obtaining a biological sample from the individual; isolatingnucleic acids from the sample; testing the biological sample for thepresence of one or more nucleic acid sequences fully complementary toone or more segments of the CFTR gene, wherein said one or more nucleicacid sequences corresponds to the mutation to be identified and isselected from the group consisting of SEQ ID NOs: 1-3; and determiningfrom the test results if the individual or the individual's offspringhas a predisposition for CF; and advising the individual accordingly. 9.The method according to claim 1, wherein the individual is of Africanorigin.
 10. The method according to claim 1, wherein the biologicalsample contains DNA.
 11. One or more nucleic acid sequences fullycomplementary to one or more segments of the CFTR gene, wherein said oneor more nucleic acid sequences correspond to one or more mutations inthe CFTR gene selected from the group consisting of SEQ ID NOs: 1-3. 12.A kit for identifying mutations in the CFTR gene of an individual,wherein the kit is provided with primers and probes required to detectthe presence of one or more nucleic acids fully complementary to one ormore segments of the CFTR gene, with the said one or more nucleic acidsequences corresponding to one or more mutations in the CFTR geneselected from the group consisting of SEQ ID NOs: 1-3.
 13. The methodaccording to claim 7, wherein the individual is of African origin. 14.The method according to claim 8, wherein the individual is of Africanorigin.
 15. The method according to claim 7, wherein the biologicalsample contains DNA.
 16. The method according to claim 15, wherein thebiological sample is any sample obtained from the individual's blood,serum, plasma, urine, skin, or hair.
 17. The method according to claim8, wherein the biological sample contains DNA.
 18. The method accordingto claim 17, wherein the biological sample is obtained from theindividual's blood, serum, plasma, urine, skin, or hair.
 19. The methodaccording to claim 10, wherein the biological sample is obtained fromthe individual's blood, serum, plasma, urine, skin, or hair.